The culture of hepatocytes represents a valuable approach for studying specific mechanisms of xenobiotic metabolism, chemical carcinogenesis, as well as pathology related to inherent or induced metabolic or biochemical abnormalities. Cultures of human hepatocytes can provide a more relevant view of human liver metabolism and disease processes than data obtained from hepatocyte cultures derived from other animal species.
Several problems have hindered in vitro studies of human hepatocytes. One problem is the availability of viable, normal adult human liver. In the past several years we have been involved with the development of an immediate autopsy program which has allowed us to obtain adult human liver tissue within 1 hr following death from patients free of liver disease.
Another problem associated with culture of human hepatocytes, as with most differentiated cells, is that these cells rarely divide in culture. By modifying a serum-free culture medium that was shown to support longterm multiplication of liver epithelial cells from Rhesus monkeys, we have developed replicative cultures of adult human liver cells. However, as with all normal cells, these liver epithelial cells eventually undergo senescence in culture.
The present invention is related to serum-less culture medium (denatured serum) for growth of continuous and non-continuous cell lines. More particularly the present invention is related to culture medium for liver epithelial cells with extended life spans.
The human liver is one of the few organs in adult man capable of regeneration. However, cultures of adult hepatocytes have never been adequately established. Those that have been established are only viable for a limited period of time and are produced in insufficient quantities for research in pharmacology, chemotherapy or oncogenesis.
There are several examples of animal liver cell cultures derived from experimental laboratory animals such as rats (Tsao et. al., Exp. Cell Res. 154: 38-52 (1984); Enat et al., "Proc. Nat. Acad. Sci. USA" 87: 1411-1415 (1984)) but these are not suitable for long term studies due to the limited life span of the culture.
Rat liver cells have been transformed by transfection with SV40 DNA (Woodworth et al, Cancer Res. 46: 4018-4026 (1987); Ledley et al., "Proc. Nat. Acad. Sci. USA" 84: 5335-5339 (1987)) but they are not suitable for human drug metabolism or carcinogenesis studies because of xenobiotic metabolism differences between rat and human liver cells. Further Woodworth reports that immortalized cell lines did not arise spontaneously. Woodworth points out that exposure to hormones and mitogenic factors, in particular EGF, insulin and glucogan, serum factors and virus infection stimulate hepatocyte DNA synthesis.
Clonally-derived cultures of human hepatocytes have been reported (Kaighn and Prince, Proc. Nat. Acad. Sci., 68, 2396-2400 (1971)), but no new data has been generated to support or refute these observations. In addition the medium used contained 17% serum. Several studies have shown that serum (Hashi and Cart J. Cell Physiol., 125, 82-90 (1985)), and more specifically transforming growth factor-beta (TGF-.beta.) present in serum (Nakaruma et al., Biochem Biophys Res Commu., 133 1042-50 (1985); Lin et al., Biochem. Biophys. Res. Commu., 143, 26-30 (1987); and Strain et al., Biochem. Biophys. Res. Commu., 145, 436-442 (1987)) cause a marked decrease in DNA synthesis of rat hepatocytes in culture.
Rat liver epithelial cells from adult rat liver tissue have been established using serum free medium (Chessebeuf and Padieu In Vitro, 20, 780-795 (1984); Enat et al. Proc. Natl. Acad. Sci., 81, 1411-1415, (1984)).
Human hepatoma cell lines have been cultured and are available (e.g. Knowles et al., U.S. Pat. No. 4,393,133, Jul. 12, 1983, Human Hepatoma Derived Cell Line) but, are not usable in carcinogenesis studies because they are tumorigenic. They were also cultured in medium containing serum.
As is known in the art, the field of tissue culture medium is empirical in nature, and there is little or no predictability as to whether conditions developed for one species will be operable in another.